Factorial chemical libraries

ABSTRACT

A library for determining the sequence of monomers in a polymer which is complementary to a receptor includes groups of pooled polymer products; wherein each pool is ordered such that a monomer sequence which binds to a receptor can be identified from the pool order in the library.

This application is a National Stage application under 35 U.S.C. § 371of PCT/US93/04145 filed Apr. 28, 1993, which claims priority to U.S.application Ser. No. 07/876,792 filed Apr. 29, 1992, now U.S. Pat. No.5,541,061.

BACKGROUND OF THE INVENTION

The present invention relates to the field of polymer screening. Morespecifically, in one embodiment the invention provides an improvedpolymer library and method of using the library to identify a polymersequence that is complementary to a receptor.

Many assays are available for measuring the binding affinity ofreceptors and ligands, but the information which can be gained from suchexperiments is often limited by the number and type of ligands which areavailable. Small peptides are an exemplary system for exploring therelationship between structure and function in biology. When the twentynaturally occurring amino acids are condensed into peptides they form awide variety of three-dimensional configurations, each resulting from aparticular amino acid sequence and solvent condition. The number ofpossible pentapeptides of the 20 naturally occurring amino acids, forexample, is 20⁵ or 3.2 million different peptides. The likelihood thatmolecules of this size might be useful in receptor-binding studies issupported by epitope analysis studies showing that some antibodiesrecognize sequences as short as a few amino acids with high specificity.

Prior methods of preparing large numbers of different oligomers havebeen painstakingly slow when used at a scale sufficient to permiteffective rational or random screening. For example, the “Merrifield”method, described in Atherton et al., “Solid Phase Peptide Synthesis,”IRL Press, (1989), incorporated herein by reference for all purposes,has been used to synthesize peptides on a solid support such as pins orrods. The peptides are then screened to determine if they arecomplementary to a receptor. Using the Merrifield method, it is noteconomically practical to screen more than a few peptides in a day.

Similar problems are encountered in the screening of other polymershaving a diverse basis set of monomers. For example, various methods ofoligonucleotide synthesis such as the phosphate-triester method and thephosphotriester method, described in Gait, “Oligonucleotide Synthesis,”IRL Press, (1990), incorporated herein by reference for all purposes,have similar limitations when it is desired to synthesize many diverseoligonucleotides for screening.

To screen a larger number of polymer sequences more advanced techniqueshave been disclosed. For example, Pirrung et al., WO 90/15070 U.S. Pat.No. 5,143,854, incorporated herein by reference for all purposes,describes a method of synthesizing a large number of polymer sequenceson a solid substrate using light directed methods. Dower et al., U.S.application Ser. No. 07/762,522, also incorporated by reference hereinfor all purposes, describes a method of synthesizing a library ofpolymers and a method of use thereof. The polymers are synthesized onbeads, for example. A first monomer is attached to a pool of beads.Thereafter, the pool of beads is divided, and a second monomer isattached. The process is repeated until a desired, diverse set ofpolymers is synthesized.

Other methods of synthesizing and screening polymers have also beenproposed. For example, Houghten et al., “Generation and Use of SyntheticPeptide Combinatorial Libraries for Basic Research and Drug Discovery,”Nature (1991) 354:84-86, discuss a method of generating peptidelibraries that are used for screening the peptides for biologicalactivity (see also, Houghton et al., “The Use of Synthetic PeptideCombinational Libraries for the Identification of Bioactive Peptides,”Peptide Research (1992) 5:351-358). Houghten synthesized a peptidecombinatorial library (SPCL) composed of some 34×10⁶ hexapeptides andscreened it to identify antigenic determinants that are recognized by amonoclonal antibody. Furka et al., “General Method for Rapid Synthesisof Multicomponent Peptide Mixtures,” Int. J. Peptide Protein Res. (1991)37:487-493, discusses a method of synthesizing multicomponent peptidemixtures. Furka proposed pooling as a general method for the rapidsynthesis of multicomponent peptide mixtures and illustrated itsapplication by synthesizing a mixture of 27 tetrapeptides and 180pentapeptides. Lam et al., “A new type of synthetic peptide library foridentifying ligand-binding activity,” Nature (1991) 354:82-84 usedpooling to generate a pentapeptide bead library that was screened forbinding to a monoclonal antibody. Blake et al. “Evaluation of PeptideLibraries: An Interactive Strategy To Analyze the Reactivity of PeptideMixtures With Antibodies,” Bioconjugate Chem. (1992) 3:510-513 discussesthe screening of presumed mixtures of 50,625 tetrapeptides and16,777,216 hexpeptides to select epitopes recognized by specificantibodies.

Lam's synthetic peptide library consists of a large number of beads,each bead containing peptide molecules of one kind. Beads that bind atarget (e.g., an antibody or strepavidin) are rendered colored orfluorescent. Lam reports that several million beads distributed in 10-15petri dishes can be screened with a low-power dissecting microscope inan afternoon. Positive beads are washed with 8M guanidine hydrochlorideto remove the target protein and then sequenced. The 100-200 μm diameterbeads contain 50-200 pmol of peptide, putatively well above their 5 pmolsensitivity limit. Three pentapeptide beads were sequenced daily. Theessence of Lam's method is that the identity of positive beads isestablished by direct sequencing.

Houghton et al. use a different approach to identify peptide sequencesthat are recognized by an antibody. Using the nomenclature describedherein, Houghton et al. screened an X₆X₅X_(4p)X_(3p)X_(2p)X_(1p) libraryand found that the mixture DVX_(4p)X_(3p)X_(2p)X_(1p) had the greatestpotency in their inhibition assay. Houghton et al. then synthesized aDVX₄X_(3p)X_(2p)X_(1p) library and identified the most potent amino acidin the third position. After three more iterations, they found thatDVPYDA (SEQ ID NO: 1) binds to the antibody with a K_(d) of 30 nM. Theessence of Houghten's method is recursive retrosynthesis, in which thenumber of pooled positions decreases by one each iteration.

Blake et al. used a “bogus coin strategy” to guide them to a preferredamino acid sequence. In this strategy a basis set of monomers (15 aminoacids) is first divided into three groups. Blake et al. chose A, L, V,F, Y (subgroup α), G, S, P, D, E (subgroup β), and K, R, H, N, Q(subgroup γ). By adjusting the “weighting” of the subgroups at eachposition in the polymer sequence, and then testing the activity of theweighted polymer against an unweighted polymer, one subgroup wasselected for each monomer position in the sequence. In an experimentconducted by Blake et al., a complete collection of tetramersX_(1p)X_(2p)X_(3p)X_(4p) was reduced to α₁α_(2γ) ₃α₄ by four inhibitionexperiments. Then the subgroups α and γ were each further subdividedinto three groups of amino acid which were used to synthesize four morecollections of weighted polymers. Inhibition studies with each of thesecollections suggested an epitope (F or Y)₁ (A or L)₂ (K or R)₃ (F orY)₄. One more iteration gave the desired epitope FLRF(SEQ ID NO: 2).

While meeting with some success, prior methods have also met withcertain limitations. For example, it is sometimes desirable to avoid theuse of the equipment necessary to conduct light directed techniques.Also, some prior methods have not produced the desired amount ofdiversity as efficiently as would be desired.

From the above, it is seen that an improved method and apparatus forsynthesizing a diverse collection of chemical sequences is desired.

SUMMARY OF THE INVENTION

An improved polymer library and method of screening diverse polymers isdisclosed. The system produces libraries of polymers in an efficientmanner, and utilizes the libraries for identification of the monomersequence of polymers which exhibit significant binding to a ligand.

According to one aspect of the invention, a library of polymers isformed using “pooled” and “unpooled” (or “separate”) coupling steps. Inthe pooled steps, each of the monomers from a basis set of monomers iscoupled to the terminus of a growing chain of monomers on a plurality ofpreviously mixed solid substrates. The mixed substrates are divided forcoupling of each individual monomer in a basis set. In separate steps,the substrates are not intermixed from a previous coupling step, andeach of the monomers in a basis set is separately coupled to theterminus of a growing chain of monomers on a plurality of the unmixedsubstrates.

According to one preferred aspect of the invention, pooled steps andunpooled steps are ordered such that the identification of a monomersequence which binds to a receptor can be readily identified from thelibrary. For example, according to one preferred embodiment of theinvention, several groups of products are derived from the synthesissteps. Each group is used to identify, the monomer at a specificposition in the polymer chain.

According to most preferred aspects of the invention, the library isconstructed using an ordered series of coupling steps in which productsresulting from a separate step are, thereafter, only subjected to pooledcoupling steps. Products resulting from a pooled coupling step whichhave not been previously subjected to an unpooled step are alwaysdivided for pooled and unpooled coupling. This ordered series of stepsresults in a relatively small number of coupling steps, but still allowsfor identification of the monomer sequence of a polymer which iscomplementary to a receptor of interest. For example, a first group ofproducts is used to identify the monomer at a first location in apolymer that is complementary to a receptor. A second group of productsis used to identify the monomer at a second location in a polymer thatis complementary to a receptor.

Accordingly, in one embodiment of the invention provides a polymerlibrary screening kit. The kit includes families of polymersX₃-X_(2p)-X_(1p), X_(3p)-X₂-X_(1p), and X_(3p)-X_(2p)-X₁ whereinX_(p3)-X_(2p)-X₁ comprises a collection of at least first and secondpolymer mixtures, the first polymer mixture having a first monomer in afirst position of polymer molecules therein, and different monomers insecond and third positions of the polymer molecules therein, and whereinthe second polymer mixture has a second monomer in the first position ofpolymer molecules therein, and different monomers in second and thirdpositions of the polymer molecules therein; X_(3p)-X₂-X_(1p) comprises acollection of at least third and fourth polymer mixtures, the thirdpolymer mixture having a third monomer in the second position and thefourth polymer mixture having a fourth monomer in the second position,each of the third and fourth polymer mixtures having different monomersin the first and third positions; and X₃-X_(2p)-X_(1p) comprises acollection of at least fifth and sixth polymer mixtures, the fifthpolymer mixture having a fifth monomer in the third position and thesixth polymer mixture having a sixth monomer in the third position, eachof the fifth and sixth polymer mixtures having different monomers in thefirst and second positions, wherein the first, third, and fourthmonomers are the same or different and the second, fourth, and fifthmonomers are the same or different.

A method of identifying first and second monomers in a polymer that iscomplementary to a receptor is also provided. The method includes thesteps of coupling first and second monomers in a first basis set toindividual substrates and mixing substrates to form first pooledproducts; coupling the first and second monomers from the first basisset to individual substrates, and not mixing the substrates to form atleast first and second separate products; separately coupling first andsecond monomers from a second basis set to substrates from the firstpooled products and not mixing the substrates to form at least third andfourth separate products, the second basis set being the same ordifferent than the first basis set; coupling the first and secondmonomers from the second basis set to individual substrates from thefirst separate products and mixing the substrates to form second pooledproducts; coupling the first and second monomers from the second basisset to individual substrates from the second separate products to formthird pooled products; and exposing a receptor to the third and fourthseparate products to identify a second monomer in a polymer which iscomplementary to a receptor, and exposing the second and third pooledproducts to the receptor to identify a first monomer in a polymer whichis complementary to a receptor.

A polymer screening technique using factoring is also disclosed.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b are schematic diagrams of specific embodiments of theinvention;

FIG. 2 illustrates a simple reaction graph;

FIG. 3 illustrates a reaction graph with pooled and separate products;

FIG. 4 illustrates a simplified reaction graph;

FIGS. 5 a, 5 b, and 5 c illustrate a family of pooled syntheses;

FIG. 6 illustrates a reaction graph for forming the productsX_(3p)X₂X_(1p);

FIG. 7 illustrates a reaction graph for all 64 trinucleotides;

FIG. 8 illustrates the synthesis of AAT, TGC, TGT, GTA, GTG, and CCG;

FIG. 9 provides an alternative representation of the invention;

FIGS. 10 a, 10 b, and 10 c illustrate a recursive retrosynthesisembodiment of the invention;

FIGS. 11 a, 11 b, and 11 c illustrate a combinational synthesis chamberof the invention; and

FIG. 12 illustrates a polymer library according to one embodiment of theinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS CONTENTS

-   -   I. Terminology    -   II. Overall Description    -   III. Polynomial Fractioning Applied to Screening    -   IV. Conclusion        I. Terminology

Ligand: A ligand is a molecule that is recognized by a particularreceptor. Examples of ligands that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleicacids, oligosaccharides, proteins, and monoclonal antibodies.

Monomer: A member of the set of small molecules which are or can bejoined together to form a polymer. The set of monomers includes but isnot restricted to, for example, the set of common L-amino acids, the setof D-amino acids, the set of synthetic and/or natural amino acids, theset of nucleotides and the set of pentoses and hexoses, as well assubsets thereof. The particular ordering of monomers within a polymer isreferred to herein as the “sequence” of the polymer. As used herein,monomers refers to any member of a basis set for synthesis of a polymer.For example, dimers of the 20 naturally occurring L-amino acids form abasis set of 400 monomers for synthesis of polypeptides. Different basissets of monomers may be used at successive steps in the synthesis of apolymer. Furthermore, each of the sets may include protected memberswhich are modified after synthesis. The invention is described hereinprimarily with regard to the preparation of molecules containingsequences of monomers such as ammo acids, but could readily be appliedin the preparation of other polymers. Such polymers include, forexample, both linear and cyclic polymers of nucleic acids,polysaccharides, phospholipids, and peptides having either α-, β-, orω-amino acids, heteropolymers in which a known drug is covalently boundto any of the above, polynucleotides, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, or other polymerswhich will be apparent upon review of this disclosure. Such polymers are“diverse” when polymers having different monomer sequences are formed atdifferent predefined regions of a substrate. Methods of cyclization andpolymer reversal of polymers which may be used in conjunction with thepresent invention rare disclosed in copending application Ser. No.796,727, filed Nov. 22, 1991 entitled “POLYMER REVERSAL ON SOLIDSURFACES,” incorporated herein by reference for all purposes. The“position” of a monomer in a polymer refers to the distance, by numberof monomers, from a terminus or other reference location on a polymer.

Peptide: A polymer in which the monomers are alpha amino acids and whichare joined together through amide bonds, alternatively referred to as apolypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are often two or more amino acid monomerslong, and often more than 20 amino acid monomers long. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemistry, Third Ed., 1988,which is incorporated herein by reference for all purposes.

Receptor: A molecule that has an affinity for a given ligand. Receptorsmay be naturally-occurring or manmade molecules. Also, they can beemployed in their unaltered state or as aggregates with other species.Receptors may be attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. Examples ofreceptors which can be employed by this invention include, but are notrestricted to, antibodies, cell membrane receptors, monoclonalantibodies and antisera reactive with specific antigenic d-terminals(such as on viruses, cells or other materials), drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles. Receptors are sometimesreferred to in the art as anti-ligands. As the term receptors is usedherein, no difference in meaning is intended. A “Ligand Receptor Pair”is formed when two macromolecules have combined through molecularrecognition to form a complex.

Specific examples of receptors which can be investigated by thisinvention include but are not restricted to:

-   -   a) Microorganism receptors: Determination of ligands which bind        to receptors, such as specific transport proteins or enzymes        essential to survival of microorganisms, is useful in a new        class of antibiotics. Of particular value would be antibiotics        against opportunistic fungi, protozoa, and those bacteria        resistant to the antibiotics in current use.    -   b) Enzymes: For instance, the binding site of enzymes such as        the enzymes responsible for cleaving neurotransmitters;        determination of ligands which bind to certain receptors to        modulate the action of the enzymes which cleave the different        neurotransmitters is useful in the development of drugs which        can be used in the treatment of disorders of neurotransmission.    -   c) Antibodies: For instance, the invention may be useful in        investigating the ligand-binding site on the antibody molecule        which combines with the epitope of an antigen of interest;        determining a sequence that mimics an antigenic epitope may lead        to the development of vaccines of which the immunogen is based        on one or more of such sequences or lead to the development of        related diagnostic agents or compounds useful in therapeutic        treatments such as for autoimmune diseases (e.g.. by blocking        the binding of the “self” antibodies).    -   d) Nucleic Acids: Sequences of nucleic acids may be synthesized        to establish DNA or RNA binding sequences.    -   e) Catalytic Polypeptides: Polymers, preferably polypeptides,        which are capable of promoting a chemical reaction involving the        conversion of one or more reactants to one or more products.        Such polypeptides generally include a binding site specific for        at least one reactant or reaction intermediate and an active        functionality proximate to the binding site, which functionality        is capable of chemically modifying the bound reactant. Catalytic        polypeptides and others are described in, for example, PCT        Publication No WO 90/05746, WO 90/05749, and WO 90/05785, which        are incorporated herein by reference for all purposes.    -   f) Hormone receptors: For instance, the receptors for insulin        and growth hormone. Determination of the ligands which bind with        high affinity to a receptor is useful in the development of, for        example, an oral replacement of the daily injections which        diabetics must take to relieve the symptoms of diabetes, and in        the other case, a replacement for the scarce human growth        hormone which can only be obtained from cadavers or by        recombinant DNA technology. Other examples are the        vasoconstrictive hormone receptors; determination of those        ligands which bind to a receptor may lead to the development of        drugs to control blood pressure.    -   g) Opiate receptors: Determination of ligands which bind to the        opiate receptors in the brain is useful in the development of        less-addictive replacements for morphine and related drugs.

Substrate or Solid Support: A material having a surface and which issubstantially insoluble in a solution used for coupling of monomers to agrowing polymer chain. Such materials will preferably take the form ofsmall beads, pellets, disks or other convenient forms, although otherforms may be used. A roughly spherical or ovoid shape is preferred.

Basis Set: A group of monomers that is selected for attachment to asolid substrate directly or indirectly in a given coupling step.Different basis sets or the same basis sets may be used from onecoupling step to another in a single synthesis.

Synthetic: Produced by in vitro chemical or enzymatic synthesis. Thesynthetic libraries of the present invention may be contrasted withthose in viral or plasmid vectors, for instance, which may be propagatedin bacterial, yeast, or other living hosts.

Symbols

-   -   x_(i) denotes the set of monomer units in reaction round i.    -   x_(ij) denotes the j'th monomer unit in reaction round i; x_(ij)        can be a null (Ø).    -   S_(i) refers to the separated products after reaction round i.    -   P_(i) refers to the pooled products of round i and all preceding        rounds.    -   X_(jp) denotes the pooling of reactants of round j only        Reaction Graphs

A filled circle ● denotes a reaction product terminating in a particularmonomer unit x_(ij). The set of reaction products terminating in x_(i)is shown by a set of circles on the same horizontal level.

Filled circles that react with each other are connected by straightlines. Pooling is shown by lines meeting below in an open circle.

A factorable polynomial synthesis is one in which each monomer unit of around is joined to each monomer of the preceding round. In a graph ofsuch a synthesis, each filled circle at one level is connected to eachfilled circle of the level above. For example, the reaction graphcorresponding to a three-round factorable synthesis with

 X₁=X₂=X₃={A,T,G,C}

which yields all 64 trinucleotides, is shown in FIG. 7.

In contrast, in an irreducible (prime) polynomial synthesis, at leastone line in the graph of the corresponding factorable polynomialsynthesis is missing. In the synthesis of AAT, TGC, TGT, GTA, GTG, andCCG only, such syntheses are illustrated in FIG. 8.

II Overall Description

FIG. 1 is an overall illustration of one aspect of the invention. Asshown therein, monomers A and B, which form all or part of a first basisset of monomers, are coupled to substrates 2 in vessels 4 a and 4 b. Thesubstrates in each of the vessels 4 a and 4 b are divided. A portion ofthe substrates from each of vessels 4 a and 4 b are mixed in vessel 6,and divided for a subsequent coupling step into vessels 6 a and 6 b.Another fraction of the monomers from vessels 4 a and 4 b is not mixed,as indicated by vessels 10 and 12.

Thereafter, the substrates are coupled to monomers from a second basisset C,D, which may or may not be the same as the basis set A,B. Asshown, the monomer C is coupled to the mixed or “pooled” substrates invessel 6 a, while the monomer D is coupled to the “pooled” substrates invessel 6 b. A portion of the products of these reactions may be mixedfor later coupling steps, but at least a portion of the products invessels 6 a and 6 b are not mixed.

The products in vessels 10 and 12 are preferably each divided forcoupling to monomer C as shown in vessels 10 b and 12 b, while thesubstrates in vessels 10 a and 12 a are used to couple the monomer D tothe growing polymer chain. The products of the reactions in vessels 10 aand 10 b are mixed or pooled, and placed in vessel 20. The products ofthe reactions in vessels 12 a and 12 b are mixed or pooled, and placedin vessel 22.

The products in vessels 20 and 22 are, thereafter, used to identify afirst monomer in a polymer which is complementary to a receptor ofinterest. It is assumed for the sake of illustration herein that themonomer sequence AC is complementary to the receptor R. A receptorlabeled with, for example, a fluorescent or radioactive label * isexposed to the materials in vessels 20 and 22, and unbound receptor isseparated from the solid supports. Binding to the substrates will occuronly with the substrates in vessel 20. Fluorescence is, therefore,observed only in vessel 20. From this observation, it is possible toconclude that the first monomer in a complementary receptor is A, sinceall of the polymers in vessel 22 contain the first monomer B.Conversely, all of the polymers in vessel 20 contain the first monomerA.

The labeled receptor is also exposed to the polymers in vessels 26 a and26 b. In this case, binding of the labelled receptor will be observedonly in vessel 26 a. Accordingly, it is possible to identify the secondmonomer in a complementary sequence as C, since none of the polymers invessel 26 b contain the second monomer C, while all of the polymers invessel 26 a contain the second monomer C. Therefore, it is possible toconclude that the sequence AC is complementary to R since binding isobserved in vessels 26 a and 20.

FIG. 1 b illustrates aspects of a preferred embodiment of the inventionin greater detail with a larger polymer chain. According to theembodiment shown in FIG. 1 b, a basis set of 3 monomers, A, B, and C isused in each coupling step. The synthesized polymers are to be threemonomers long. It will be recognized by those of skill in the art thatthe number of monomers in a basis set and the number of coupling stepswill vary widely from one application to another. Also, interveningcoupling steps of, for example, common monomer sequences may be used insome embodiments. Therefore, when a polymer is represented by, forexample, the notation “ABC” or “ABE” herein, it is to be understood thatother common monomers may be added such that ABDC and ABDE arerepresented by ABC and ABE. The embodiment shown in FIG. 1 b is providedmerely as an illustration of the invention.

As shown in FIG. 1 b, the synthesis takes place on a plurality ofsubstrates 2. According to a preferred aspect of the invention, thesubstrates 2 take the form of beads, such as those made of glass,resins, plastics, or the like. The term “beads” is used interchangeablyherein with the word “substrate,” although it is to be understood thatthe beads need not take on a circular or ovoid shape and can take theform of any suitable substrate. It will be further understood that thesubstrates 2 are shown only in the top portion of FIG. 1 b, but thesubstrates will be present in each of the reaction products shown inFIG. 1 b to the left of the monomer sequences. In each vessel in FIG. 1b, all of the possible polymer products are listed. Many “copies” ofeach sequence will generally be present.

According to one embodiment, conventional Merrifield techniques are usedfor the synthesis of peptides, such as described in Atherton et al.,“Solid Phase Peptide Synthesis,” IRL Press, (1989), previouslyincorporated herein by reference for all purposes. Of course othersynthesis techniques will be suitable when different monomers are used.For example, the techniques described in Gait et al., OligonucleotideSynthesis, previously incorporated by reference herein by reference forall purposes, will be used when the monomers to be added to the growingpolymer chain are nucleotides. These techniques are only exemplary, andother more advanced techniques will be used in some embodiments such asthose for reversed and cyclic polymer synthesis disclosed in U.S.application Ser. No. 07/796,727, previously incorporated herein byreference for all purposes.

A large number of beads are utilized such that the beads may beseparated into separate reaction vessels in later steps and still bepresent in sufficient numbers such that the presence of a complementaryreceptor may be detected. As a general rule, it will be desired to use10 to 100 or more times the number of combinational possibilities forthe synthesis so as to ensure each member of each set is synthesized.Also, the use of a large number of beads ensures that pooled reactionproducts are distributed to each succeeding reaction vessel when apooled group of beads is divided.

The beads are preferably as small as possible so that the reactionvessels and other material handling equipment utilized in the processmay also be as small as possible. Preferably, the beads have a diameterof less than about 1 mm, and preferably less than about 100 μm, and morepreferably less than about 10 μm. In some embodiments, the synthesis iscarried out in solution. In other embodiments, the synthesis is carriedout on solid substrates, and the resulting polymers are then cleavedfrom the substrates before binding with a receptor.

As shown in FIG. 1 b the monomers A, B, and C are coupled to substratesin three reaction vessels 4 a, 4 b, and 4 c, respectively. A singlesubstrate is shown in FIG. 1 b for purposes of clarity, but it will berecognized that in each reaction vessel a large number of beads will bepresent. Accordingly, a large number of “copies” of the substrates withthe respective monomers coupled thereto are formed in each of reactionvessels 4 a, 4 b, and 4 c. It will be recognized that the monomers neednot be directly coupled to the substrate, and in most cases linkermolecules will be provided between the monomers and the substrate, suchas those described in U.S. application Ser. No. 07/624,120, incorporatedherein by reference for all purposes. Also, it should be recognized thatthe steps shown in FIG. 1 b may be preceded by or followed by othersynthesis steps which may or may not be combinational steps using thetechniques described herein.

Thereafter, a fraction of the products in each of vessels 4 a, 4 b, and4 c are combined, mixed, and redistributed to each of reaction vessels 6a, 6 b, and 6 c. The remaining fraction of the products in each ofvessels 4 a, 4 b, and 4 c is not combined. Instead, the remainingfraction of the products in reaction vessel 4 a is divided and placed inreaction vessels 8 a, 8 b, and 8 c. Similarly, the remaining fraction ofthe products in vessel 4 b is divided and placed in vessels 10 a, 10 b,and 10 c. The remaining fraction of the products in reactant vessel 4 cis divided and placed in reaction vessels 12 a, 12 b, and 12 c.

The reactants placed in vessels 6 a, 6 b, and 6 c are referred to hereinas “pooled” reactants since they comprise a mixture of the productsresulting from the previous coupling step. The reactants placed invessels 8, 10, and 12 by contrast are separate reactants since they arenot mixtures of the products from the previous coupling steps. Accordingto a preferred embodiment of the invention, after the reactants invessels 8, 10, and 12 are subjected to a separate coupling step, theyare subjected only to pooled coupling steps thereafter. Conversely, ineach subsequent coupling step, the pooled reactants are subjected to acoupling step, and divided for subsequent separate and pooled couplingsteps.

Preferably, the reactants are divided such that a greater fraction ofthe beads is distributed for pooled synthesis. For example, in FIG. 9, ⅘of the beads would go to the first pooled group 905 while ⅕ would go tothe unpooled group 903.

Thereafter the monomers A, B, and C are coupled to the growing polymerchain in reaction vessels 8 a, 8 b, and 8 c, respectively. The resultingpolymers then have the monomer sequence CA, CB, and CC in reactionvessels 8 a, 8 b, and 8 c, respectively. The products of these reactionsare then mixed or pooled in reaction vessel 9, and the mixture is againdivided among reaction vessels 14 a, 14 b, and 14 c. The monomers A, B,and C are again coupled to the growing polymer chains in vessels 14 a,14 b, and 14 c, respectively. The products of these reactions are againmixed or pooled and placed in vessel 16 a.

Similarly, the monomers A, B, and C are coupled to the growing polymerchain in reaction vessels 10 a, 10 b, and 10c, then mixed in vessel 18,divided, and placed in reaction vessels 20 a, 20 b, and 20 c. MonomersA, B, and C are coupled to the growing polymer chain in vessels 20 a, 20b, and 20 c respectively, mixed, and placed in vessel 16 b. Monomers A,B, and C are also coupled to the growing polymer chain in reactantvessels 12 a, 12 b, and 12 c respectively, mixed, and placed in vessel21. These products are divided for reaction with monomers A, B, and C invessels 22 a, 22 b, and 22 c respectively, mixed, and placed in vessel16 c. A characteristic feature of the preferred embodiments of thepresent invention should be noted in the right half of FIG. 1 b.Specifically, once the products of a reaction are not pooled (such as invessels 8, 10, and 12), the products of coupling steps are always pooledthereafter.

Referring to the left hand portion of FIG. 1 b, the pooled reactants invessels 6 a, 6 b, and 6 c are coupled to monomers A, B, and Crespectively, resulting in the products shown in vessels 26 a, 26 b, and26 c. Since the products in vessels 26 a, 26 b, and 26 c are derivedfrom a “chain” of pooled reactions, the products are separated for bothpooled and separate reactions Specifically, a portion of the substratesin vessels 26 a, 26 b, and 26 c are combined, mixed, and divided forpooled reactions with monomers A, B, and C in vessels 28 a, 28 b, and 28c respectively In addition, the remaining portion of the products invessels 26 a , b, and c are separately divided and placed in reactionvessels 30 a-c, 32 a-c, and 34 a-c respectively. The materials invessels 30 a, 32 a, and 34 a are coupled to monomer A, the materials invessels 30 b, 32 b, and 34 b are coupled to monomer B, and the materialsin vessels 30 c, 32 c, and 34 c are coupled to monomer C. Since theproducts in vessels 30, 32, and 34 result have been preceded by aseparate reaction, the products in vessels 30, 32, and 34 are pooled, ormixed, and placed in vessels 36 a, 36 b, and 36 c, respectively.

For reasons that will be discussed further below, the vessels in group42 are used to determine the identity of the monomer in the firstposition in a polymer that is complementary to a receptor. The vesselsin group 44 are used to determine the identity of the second monomer ina polymer that is complementary to a receptor. The vessels in group 46are used to determine the identity of the third monomer in a polymerthat is complementary to a receptor.

For example, assume that a given receptor is complementary to themonomer sequence ABC, but the sequence of the complementary polymer isnot known ab initio. If the receptor is labelled with an appropriatelabel such as fluorescein and placed in each of the vessels in groups42, 44, and 46, fluorescence will be detected only in vessels 16 c, 36b, and 28 c since the polymer sequence ABC appears only in thesevessels. Fluorescence may be detected using, for example, the methodsdescribed in Mathies et al., U.S. Pat. No. 4,979,824, incorporatedherein in its entirety by reference for all purposes.

Since all of the polymers in vessel 16 c have monomer A in the firstposition, and none of the polymers in vessels 16 a or 16 b have monomerA in the first position, it is readily determined that the monomer inthe first position of a complementary polymer is the monomer A.Similarly, since all of the polymers in vessel 36 b have the monomer Bin the second position, it is readily determined that the monomer B mustoccupy the second position of a complementary polymer sequence.Similarly, since all of the polymers in vessel 28 c have a C monomer inthe third position, the complementary receptor must have a C in itsthird position. Therefore, it would readily be determined that thecomplementary sequence to the receptor has the monomer sequence ABC.

As will be seen upon careful examination of the sequences in the vesselgroups 42, 44, 46, ambiguities will generally not arise, regardless ofthe monomer sequence which is complementary to the receptor of interest.As a point of comparison, if the receptor of interest is complementaryto the sequence BBA, fluorescence would be detected only in vessels 16b, 36 b, and 28 a. From this information is becomes clear that thecomplementary monomer sequence must be BBA.

The above embodiment illustrates the synthesis of pooled groups ofpolymers by way of separation into separate vessels, followed bycoupling and mixing. It will be recognized that this is only forconvenience of illustration and that in some embodiments the pooledgroups of polymers will be synthesized under controlled conditions bysimultaneous reaction of each of the monomers to be coupled to thepolymers in a single reactor. Further, it will be recognized that thesynthesis steps above will be supplemented in many embodiments by prior,intermediate, and subsequent coupling steps, which are not illustratedfor ease of illustration.

The above method may be generally illustrated by way of the adoption ofappropriate nomenclature. For example, let X_(i) denote the set ofmonomer units that become joined to a growing chain at reaction round i.For example, suppose thatX₁={L,G}X₂={P,Y}X₃={R,A}A particular monomer is denoted by x_(ij). For example,x_(3,1)=RThe reaction products S₃ of such a three-round peptide synthesis isconcisely represented byS₃=X₃X₂X₁S₃ is determined by expanding a reaction polynomial as described inFodor et al., Science (1991) 251:767-773, incorporated herein byreference for all purposes.S ₃=(R+A)(P+Y)(L+G)and so S₃ consists of 8 tripeptides:RPL, RYL, RPG, RYG, APL, AYL, APG, and AYGS_(ij) denotes a set of reaction products terminating in monomer unitx_(ij). In the above synthesis, for example,S₁₂=GS₂₁={PL,PG}S₃₂={APL,AYL,APG,AYG}

Thus three-round synthesis can also be represented by a reaction graph,as shown in FIG. 2. Each reaction product of round i is depicted by afilled dot on the same horizontal level. Each dot of round i is joinedto each dot of the preceding round and to each dot of the succeedinground. For example, the dot denoting S₂₁ is joined to the dots for S₁₁and S₁₂, and also to the dots S₃₁ and S₃₂. Note that dots on a level arenever connected to each other because, by definition, monomer units of around do not combine with one another.

It is generally assumed that the products of each round are spatiallyseparate and addressable. Each can then be readily assayed. However, thenumber of compounds generated by a combinational synthesis can, after afew rounds, greatly exceed the number of experimentally available binsor vessels. It is then advantageous to pool the products of one or morerounds of synthesis. For example, a five-round synthesis using the basicset of 20 amino acids yields 20⁵ or 3.2×10⁶ pentapeptides. In contrast,if the products of the first two rounds are pooled, the subsequent threerounds yield only 8,000 sets of products. Information is lost in thepooling process, but the number of products becomes experimentallytractable.

The above representation of combinational synthesis may be modified totake into account the effect of pooling. Suppose that products of thefirst two rounds of the three-round synthesis mentioned earlier arepooled. The reaction graph for a with pooled steps is shown in FIG. 3.The pooled products of round i are denoted by P_(i) to distinguish themfor the separate products S_(i). In a reaction graph, pooling is shownby the convergence of lines from the S_(i) that are pooled. P₁ is thenshown as an open circle.

In this example,P ₁ ={L+G}P ₂ ={PL+PG+YL+YG}S ₃ 32 X ₃ P ₂ ={RPL+RPG+RYL+RYG,APL+APG+AYL+AYG}The plus sign joins products that are present in a mixture. In contrast,products separated by commas are located in separate bins and arespatially addressable. In this example, the pooled products of thesecond round are located in one bin, whereas the products after threerounds are located in two bins. One bin contains the mixtureRPL+RPG+RYL+RYG, and the other bin contains the mixture APL+APG+AYL+AYG.

This reaction graph can be simplified. Suppose that P₁ was coupled to aequimolar mixture of x₂₁ and x₂₂ in a single bin. If the couplingefficiencies for all species are the same, the amounts and kinds ofproducts obtained would be the same as that given by coupling P₁ withx₂₁ and x₂₂ in separate bins and then pooling the products. Thus, pooledproducts and pooled reactants are formally equivalent provided that thereactions occur in a substantially homogeneous solution and all couplingefficiencies are substantially the same. Hence, an X₃P₂ synthesis can bemost simply represented by the reaction graph shown in FIG. 4.

The line joining P₂ to P₁ means that all products in P₁ are coupledequally to all reactants X₂, either by (1) adjusting the concentrationsof reactants or (2) driving the reactions to completion in separatebins, followed by pooling. For beads or other discrete particles, (2)more often applies so that each particle expresses only one kind ofproduct.

By way of comparison, the synthesis of 180 pentapeptides in Furka etal., “General Method for Rapid Synthesis of Multicomponent PeptideMixtures,” Int. J. Peptide Protein Res. (1991) 37:487-493, isrepresented with the above nomenclature as S₅=X₅P₄, where X₁={A},X₂={E,F,K,}, X₃={E,P,K}, X₄={E,F,G,K}, and X₅={E,G,K,L,P}. The peptidecombinatorial library synthesis in Houghten et al., “Generation and Useof Synthetic Peptide Combinatorial Libraries for Basic Research and DrugDiscovery,” Nature (1991) 354-84-86 is S₆=X₆ X₅P₄, where each X₁ is aset of 18 naturally occurring amino acids. The S₆ products are locatedin 18×18 or 324 bins, each containing a mixture of 18³=5,832hexapeptides. The pooled synthesis in Lam et al., “A new type ofsynthetic peptide library for identifying ligand-binding activity,”Nature (1991) 354:82-84, is represented using the above nomenclature asP₅, where each X_(i) is a set of 19 naturally-occurring amino acids. P₅is a mixture of 19⁵=2.48×10⁶ beads, each bearing one kind of peptide.

In the pooled syntheses of Houghten, Lam, and Furka, all products fromround i to round n are mixed. In Furka's synthesis (X₅, P₄), the firstfour rounds are pooled. In an X₃P₂ synthesis, the first two rounds arepooled.

Representative pooled syntheses techniques according to one preferredembodiment of the invention herein are shown in FIGS. 5 a, 5 b, and 5 c.The symbol X_(ip) means that the reactants of round i have been pooledwithout pooling the reaction products of previous rounds. This isachieved by, for example, (1) mixing the reactants X_(i) or (2) byreacting each member of X_(i) with each reaction product of S_(i−1), asshown in FIG. 6 for X_(3p)X₂X_(1p).

For pentapeptides made of the naturally occurring 20 amino acids forexample, a family of five pooled syntheses groups according to theinvention herein will be particularly useful:X₅X_(4p)X_(3p)X_(2p)X_(1p)X_(5p)X₄X_(3p)X_(2p)X_(1p)X_(5p)X_(4p)X₃X_(2p)X_(1p)X_(5p)X_(4p)X_(3p)X₂X_(1p)X_(5p)X_(4p)X_(3p)X_(2p)X₁The products of each of these five syntheses product groups would belocated in 20 physically isolated bins. Each bin would contain adifferent mixture of 160,000 pentapeptides. As with the trimerillustrated in FIG. 1 b, the identity of the monomers forming acomplementary pentamer would be determined unambiguously by identifyingwhich of the 20 bins in each of the five syntheses product groups showedbinding to a receptor.

It is to be recognized that while “bins” are referred to herein for thesake of simplicity, any of a variety of techniques may be used forphysically separating the peptide or other polymer mixtures.

More specifically, a sequence of monomers in a complementary ligand fora receptor is identified as follows. For example, consider the family ofpooled tripeptide libraries made of the 20 naturally occurring aminoacids:X₃X_(2p)X_(1p) X_(3p)X₂X_(1p)X_(3p)X_(2p)X₁The most potent amino acid at the left position (x₃₁) is revealed byanalysis of the 20 bins of X₃X_(2p)X_(1p); x_(2j) is determined byanalysis of X_(3p)X₂X_(1p); and x_(1k) is determined by analysisX_(3p)X_(2p)X_(1p). The sequence of the most potent tripeptide is thenpredicted to be x_(3i)x_(2j)x_(1k). Accordingly, each pooled group inthe library reveals the identity of a monomer in a different position ina complementary polymer.

It will be recognized that it will not always be desirable to determinethe identity of the entire sequence of monomers in a polymer that iscomplementary to a receptor. Instead, it will only be necessary todetermine the identity of selected monomers in a polymer in someinstances. The monomers of interest may be at intermediate locations onthe chain of polymer, and may be interspersed by other monomers.Accordingly, in a more general sense, the method herein provides for thesynthesis of a library of polymers. The library is used to identify atleast two monomers of interest in the polymer chain.

For example, the identity of the x_(2j) monomer is determined byanalysis of a library of polymers T-X₂-I-X_(1p)-T; and the identity ofthe monomer x_(1k) is determined by analysis of a library of polymersT-X_(2p)-I-X₁-T, where T indicates terminal groups on the polymer chain,which may be null groups, and I designates intermediate groups in thepolymer chain, which may also be null groups.

The method of making the library used pooled and separate synthesissteps. The polymers have at least two monomer locations at which it isdesired to determine the identity of monomers which provide a polymerwith a sequence complementary to a receptor. The library is synthesizedsuch that the products of a pooled synthesis are separated and subjectedto a separate synthesis and a second pooled synthesis. The products ofthe separate synthesis are subjected to a sense of pooled syntheses,without any further separate synthesis in preferred embodiments.Conversely, the products of the second pooled synthesis are divided andsubjected to both a separate syntheses and a third pooled synthesis.

The synthesis steps result in a library of polymers having at leastfirst and second subsets. The first subset is used to determine theidentity of a monomer or monomers at a first location in the polymerchain which is complementary to a receptor. The second subset of thelibrary is used to determine the identity of a monomer or monomers at asecond location in the polymer chain which is complementary to areceptor.

The method uses summated assays to identify optimal sequences. Thedistribution of activities in the mixture assayed remains unknown. Onlythe aggregate activity is determined More information can be obtainedfrom analyses of beads or other particles that contain multiple copiesof one kind of sequence. The activity of each bead can be quantitatedeven though its identity is unknown.

Suppose that 2 μm diameter beads are used for pooled syntheses. Somepertinent properties of typical beads are:

-   -   Volume=4.2 μM³    -   Surface area=12.6 μm²    -   Number of target sites=1.3×10⁵    -   (assuming 1 per 100 mm²)    -   Number of beads per cm³=2.4×10¹¹

Fluorescence measurements of beads flowing rapidly through a laser beamare made using techniques such as those in U.S. Pat. No. 4,979,824,previously incorporated herein by reference for all purposes, whichprovide exemplary methods for determining the distribution of activitiesin a pooled synthesis.

Assume a light beam diameter of 2 μm is used for detection offluorescein labeled beads, at a flow rate of 20 cm/s. The transit timeof a bead through the beam is then 10 μs. The emission rate from asingle chromophore can be as high as 10⁸s⁻¹. If 10% of the target sitesare occupied, this corresponds to an emission rate of about 10¹²s⁻¹, or10⁷ emitted photons in 10 μs, which would be easily detected. If 10% ofthe sample volume is occupied by beads, an average of one bead wouldpass through the beam every 0.1 ms. Thus, 10⁴ beads could be analyzedper second. A library of 3.2×10⁶ beads (each bearing a differentpentapeptide) could be analyzed in about 6 minutes.

Alternatively, the beads may be analyzed by spreading them on a surface.For example, 3.2×10⁶ beads would occupy 1.28×10⁷ μm² if packed togetherin a square array. In 1.28 cm², these beads would occupy 10% of thesurface area. Smaller beads, say 0.2 μm², would give a sufficientfluorescence signal. The advantage of smaller beads is that higher beaddensities could be used, leading to a marked reduction in the timeneeded for analysis.

The fluorescence pulse height distribution emerging from either analysiswould reveal whether there are many or few optimal sequences containedwithin the sample of beads. In the simplest case, a single bright beadis seen in just one bin of a pooled synthesis. The identity of the bestsequence then comes directly from analysis of each pooled synthesis ofthe family.

In other cases, there is a distribution of intensities within severalsets of beads. As a general rule, positioned libraries where binding isexhibited in multiple bins indicates that a particular position plays aless significant role in binding. In some embodiments, positions whereambiguity are detected are further evaluated through use of the VLSIPS™technique, The VLSIPS™ arrays will vary only those positions wherein themonomer has not been determined unambiguously. The present invention isused, therefore, to reduce the number of polymers which will be screenedwith VLSIPS™ in some embodiments.

In still other cases, polymer mixtures synthesized in multiple bins arecleaved from their respective beads and then assayed for activity. Thefreed polymers are then able to interact with receptors in variousorientations. The activity of such polymers can be assayed by variouswell-known techniques such as ELISA.

FIG. 9 provides an alternative description of the invention. As showntherein, at step 901 a collection of substrates is subjected to pooledand separate coupling steps, resulting in pooled and separate products905 and 903, respectively. In comparison with the embodiment shown inFIG. 1 b, products 903 are analogous to the products shown in vessels 8,10, and 12, and products 905 are analogous to the products in vessel 6.The collection of substrate products 903 are then subjected to pooledcoupling steps 903, 905, 907, and 909, i.e., the subsequent couplingsteps to the separate reactants are only pooled coupling steps.Accordingly, the identity of the monomer in the first position of apolymer complementary to a receptor is determined by evaluation of theproducts 907.

Conversely, the pooled products 905 are divided and subjected to pooledand separate coupling steps 909, resulting in pooled and separateproducts 907 and 913, respectively. As with the separate products 903,the separate products 913 are subjected only to pooled coupling stepsthereafter, resulting in pooled products 915 and 917. The products 917are used to determine the monomer in a second position in a polymercomplementary to a receptor of interest.

In the same manner, the pooled products 907 are divided and subjected topooled and separate coupling steps 919, resulting in pooled and separateproducts 923 and 921. The separate products 923 are subjected only to apooled reaction thereafter, the products 925 being used to determine themonomer in a third position in a polymer complementary to a receptor ofinterest. The pooled products 921 are divided and subjected to pooledand separate reactions 927, resulting in pooled and separate products929 and 931. The products 907, 917, 925, 931, and 929 are used toidentify complementary receptors. In the preferred embodiment, thepooled products 927 are first used to determine if any polymers ofinterest are present. The separate products 931 are used to determinethe identity of a monomer in a fourth position of a polymercomplementary to a receptor.

As shown in FIG. 9, pooled products that have not been subjected toprior separate reactions are divided and subjected to pooled andseparate reactions according to the invention herein. Conversely,products which result from a prior separate coupling step are onlysubjected to pooled coupling steps.

In an alternative embodiment depicted in FIGS. 10 a-10 c, a recursiveretrosynthesis is employed to screen a diverse set of polymers. Unlikethe recursive retrosynthesis method of Houghten et al. described supra,this method identifies the sequence of a “best” polymer by identifying acollection of polymers (“library”) containing the bead giving thestrongest signal. Houghton et al., in contrast, identify the entirelibrary, rather than the single polymer (bead), having the strongestsignal. Thus, the technique of Houghton et al. may identify an incorrectmonomer in the sequence of interest because of the library containingthat monomer gave the strongest signal, while the “best” polymer islocated in a different library. The recursive retrosynthesis embodimentof this invention overcomes this difficulty of Houghton et al.'s byidentifying the individual polymer giving the strongest signal.

Referring to FIGS. 10 a-10 c, an example of this process is describedfor the set of all pentamers formed from a basis set of 50 monomers. Asshown in FIG. 10 a, the complete collection of quadramers is synthesizedon a number of beads (e.g., 3×10⁶ beads) by four cycles of alternatelydividing, reacting, and pooling the beads. The pool of quadramers isthen divided into 50 bins, each of which is reacted with a differentmember of the basis set to give 50 bins of pentamers, each containingonly those pentamers terminating in a specified monomer. In the notationused herein, this collection of polymers is represented byX_(1p)X_(2p)X_(4p)X₅₍₁₋₅₀₎. The individual beads in each bin are thenassayed to identify the single best bead (i.e., the bead providing thestrongest signal on binding with the receptor of interest). This may beaccomplished in about eight hours by FACS as described above, forexample. Having determined the bin containing the bead providing thestrongest signal, the identity of the monomer in the fifth position isknown. In the example of FIG. 10, that monomer is D.

Next, the complete collection of trimers is formed as before (from e.g.,6×10⁶ beads) by cycles of dividing, reacting, and pooling the beads asshown in FIG. 10 b. Alternatively, the library of trimers could be setaside after the third cycle of the previous step (during formation ofthe complete library of pentamers). At this point, the pooled beads aredivided into 50 bins, each of which is reacted with a different memberof the basis set. The quadramers in each bin are then reacted with D toproduce a collection of beads represented by X_(1p)X_(2p)X_(3p)X₄₍₁₋₅₀₎D. The beads in each bin are then assayed to again identify the beadgiving the strongest signal. The bin from which that bead was takenidentified the monomer at the next position: A in this case. The aboveprocess is repeated to produce X_(1p)X_(2p)X₃₍₁₋₅₀₎ AD and identify themonomer at the next position as shown in FIG. 10 c. In this example, thenext monomer is identified as Q. The final two monomers of the sequencecan be identified in the same fashion. However, it may be more efficientto simply screen the remaining 2,500 possible pentamers via a VLSIPS™technique.

In general, for a polymer of length N synthesized from a basis set of nmonomers, the terminal monomer may be identified by the followingprocedure. First, a pooled library of substrates is formed such thateach substrate has a different polymer synthesized. The pooled libraryincludes a collection of polymers represented by X_(1p)X_(2p) . . .X_((N-1)p). The library is divided into n separate bins, each of whichis then reacted with a different monomer to form a library X_(1p)X_(2p). . . X_((N-1)p)X_(N(1-n)). Finally, a receptor is exposed to thesubstrate in each of the n separate bins to identify the bin containingthe polymer which binds the receptor most strongly. This bin providesthe identify the monomer in the X_(N) position. The penultimate monomeris identified by a similar procedure from X_(1p)X_(2p) . . .X_((N-1)(1-n))R, where R is the terminal monomer previously identified.Each succeeding monomer can be identified in the same manner. In thisexample, the two basis sets used to identify the monomers at positions Nand N-1 each contained n members. It will be appreciated that themonomer basis sets used to identify the monomer at each position on thepolymer may independent of the other basis sets.

A combinational synthesis chamber for conducting the synthesis, pooling,and dividing steps employed in each cycle of this invention isillustrated in FIG. 11 a-11 c. Individual chambers 200, each containingan amount of packed beads 203, are aligned in close proximity to oneanother to form a two-dimensional array. The reaction chambers 200 aremounted on a base 207 via passages 211. A filter 213 is provided at thebase of each coupling chamber 200 to prevent the beads from leaving thecoupling chambers 200 when solutions are drained through passages 211.In synthesis mode, coupling solutions are introduced through passages205 while cover 201 is in a closed position (as shown in FIG. 11 a). Thecontents of each chamber 200 are prevented from contacting adjacentchambers by cover 201. Passages 205 and 211 are controlled by valves orother control mechanisms not shown.

After the coupling reactions have proceeded to the desired extent, thecoupling solutions are drained from the synthesis chamber through thepassages 211. Subsequent washing steps may be necessary before poolingand redistribution. In such washing steps, washing solutions areintroduced through passages 205 while cover 201 is held in the closedposition. After sufficient time has elapsed, the washing solution isdrained through passages 211. Multiple washing steps may be performed asnecessary to remove unused coupling solutions from chambers 200. Duringthe reacting and washing steps, the beads in the reaction chambers maybe agitated by rotating or shaking the entire synthesis chamber.

As shown in FIG. 11 b pooling and dividing of the beads is accomplishedby sliding cover 201 away from base 207 to form a mixing chamber 215.The previously packed beads 203 are then agitated in a fluid such thatthey mix within mixing chamber 215. Ultimately, when the agitation isstopped, the beads will settle randomly into various chambers 200. Atthat point, the suspension solution can be drained through passages 211,and cover 201 can be lowered to rest on the tops of chamber 200 as shownin FIG. 11 a. During the pooling and dividing stages, the valves inpassages 205 and 211 are closed. A sealing means 209 is provided toprevent beads or fluid from leaving the synthesis chamber.

FIG. 11 c shows a top view of a two-dimensional array of reactionchambers 200 mounted on base 207. The chambers shown are arranged in a7×7 array of 49 chambers.

FIG. 12 illustrates a library of polymers which will be useful inaccordance with the invention herein. As shown therein, the polymershave a number of monomer positions, designated by p_(i). The monomershave at least two positions of interest, p₁ and p₂. p₁ and p₂ may insome embodiments be separated by various intermediate monomers orgroups, and may also have various terminal groups attached thereto. Themonomers are placed in a number of physically isolated bins or vessels1002. The bins or vessels 1002 may in fact be attached, such as in amicrotiter plate, or the bins/vessels may be distinct containers such astest tubes, microtiter trays, or the like.

A first bin 1002 a contains polymers with a first monomer M₁ in thefirst position p₁ in each of the polymers therein. However, the polymermolecules in the first bin have a variety of different monomers such asM₁, M₂, and M₃ in a second position p₂. In the second bin 1002 b asecond monomer M₂ is in the first position p₁ in each of the polymerstherein, while different monomers such as M₁, M₂, and M₃ are in thesecond position p₂. In the third bin 1002 c a third monomer M₃ is in thefirst position p₁ in each of the polymers therein, while differentmonomers such as M₁, M₂, and M₃ are in the second position p₂. Thefirst, second, and third bins comprise all or part of a collection ofbins . . . X₁ . . . X_(2p) . . . .

Conversely, fourth bin 1002 d contains polymers with a first monomer M₁in the second position p₂ in each of the polymer molecules therein. Thepolymer molecules in the first bin have a variety of different monomerssuch as M₁, M₂, and M₃ in their first position p₁. In the fifth bin 1002e a second monomer M₂ is in the second position p₂ in each of thepolymers therein, while different monomers such as M₁, M₂, and M₃ are inthe first position p₁. In the sixth bin 1002 f a third monomer M₃ is inthe second position p₂ in each of the polymers therein, while differentmonomers such as M₁, M₂, and M₃ are in the first position p₁. Thefourth, fifth, and sixth bins comprise all or part of a collection ofbins . . . X_(1p) . . . X₂ . . . .

In screening studies, the bins 1002 a, 1002 b, and 1002 c are used todetermine the identity of the monomer in position 1 of a polymer that iscomplementary to a receptor of interest. The bins 1002 d, 1002 e, and1002 f are used to determine the identity of the monomer in position 2of a polymer that is complementary to a receptor of interest.

It will be recognized that the polymers which are screened according tothe above methods can be of widely varying length and composition. Forexample, in preferred embodiments, the polymer molecules are preferablygreater than 3 monomer units long, preferably greater than 5 monomerunits long, more preferably greater than 10 monomer units long, and morepreferably more than 20 monomer units long. Although a simplifiedlibrary is shown in FIG. 12, it will be recognized that in mostembodiments, the library will include additional polymer bins so as toidentify the monomers at more than 3 positions, preferably more than 5positions, more preferably more than 10 positions, and more preferablymore than 20 positions in a complementary polymer to a receptor.

III. Polynomial Factoring Applied to Screening

In some embodiments a population of all possible polymers of length nare synthesized. If a receptor is found to bind with one of the polymersin the mixture, a second synthesis is conducted in which the polymersare “factored,” i.e., two units are formed, each having half of thepopulation synthesized initially. It is then determined which of the twobins shows binding to the receptor, the bin which exhibits binding beingreferred to as a “target group.” Yet another synthesis is conducted inwhich two bins are created, each with half of the population of thetarget group in the earlier bin. The process is repeated until thesequence of the polymer or polymers that show binding to the receptorsis determined.

More specifically, the invention provides for the synthesis of apopulation:$P = {\sum\limits_{i = 1}^{n}{X_{i}{\sum\limits_{j = 1}^{n}X_{j}}}}$This solution is factored as:$P = {{\sum\limits_{i}{X_{i}\lbrack {{\overset{n/2}{\sum\limits_{j}}X_{j}} + {\overset{n}{\sum\limits_{n/2}}X_{j}}} \rbrack}} = {P_{1} + P_{2}}}$where:${P_{1} = {\sum\limits_{i = 1}^{n}{X_{i}{\sum\limits_{j = 1}^{n/2}X_{j}}}}};\quad{{{and}\quad P_{2}} = {\sum\limits_{i = 1}^{n}{X_{i}{\overset{n}{\sum\limits_{n/2}}X_{j}}}}}$

If P₁ generates a “hit,” P₁ is factored. If P₂ generates a “hit,” P₂ isfactored. Each synthesis requires only half the number of polymers madein the prior step.

IV. Conclusion

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example whilethe invention is illustrated primarily with regard to the synthesis ofoligonucleotides and peptides, the invention will also find utility inconjunction with the synthesis and analysis of a wide variety ofadditional polymers. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

1. A polymer library comprising polymers selected from the groupconsisting of oligonucleotides and peptides, wherein said librarycomprises at least three families of polymers pools, which polymers eachcomprise at least three monomers each obtained from a basis set ofmonomers, where: the polymers in a first family are represented byX₃X_(2P)X_(1P), wherein X₃ represents a first monomer position which isoccupied by a different monomer in each of then pools of polymers,X_(2P) represents a second monomer position which is occupied by amixture of monomers, and X_(1P) represents a third monomer positionwhich is occupied by a mixture of monomers; the polymers in a secondfamily are represented by X_(3P)X₂X_(1P) where X_(3P) represents a firstmonomer position which is occupied by a mixture of monomers, X₂represents a second monomer position which is occupied by a differentmonomer in each of the pools of polymers, and X_(1P) represents a thirdmonomer position which is occupied by a mixture of monomers; thepolymers in a third family are represented by X_(3P)X_(2P)X_(1P),wherein X_(3P) represents a first monomer position which is occupied bya mixture of monomers, X_(2P) represents a second monomer position whichis occupied by a mixture of monomers, and X₁ represents a third monomerposition which is occupied by a different monomer in each of the poolsof polymers; wherein each pool is ordered such that a monomer sequencewhich binds to a receptor can be identified from the order of pools inthe library; wherein said pools of polymers are bound to substrates on aknown region of a surface; and wherein each pool is physically isolatedfrom all the other pools in the same family in separate bins.
 2. Thepolymer library according to claim 1, wherein the library comprises Nfamilies of polymers of length N synthesized from a basis set of nmonomers, wherein for each additional monomer of polymer length greaterthan three, the polymers in each family as set forth in claim 1 comprisean additional residue X_(N(1-n)) representing an Nth monomer positionwhich is occupied by a mixture of monomers; and the polymer librarycomprises an additional family of pools of polymers wherein each pool ofthe family has a different terminal monomer and all other monomerpositions are occupied by a mixture of monomers.
 3. The polymer libraryof claim 1, wherein said polymers comprise at least four monomers. 4.The polymer library of claim 1, further comprising a labeled receptor,and a means for identifying which of said families of polymers binds tosaid labeled receptor.
 5. The polymer library of claim 4, wherein saidreceptor is labeled with a fluorescein label.
 6. The polymer libraryaccording to claim 1, wherein said substrates are beads.
 7. The polymerlibrary of claim 6, wherein said beads are spread on an array.
 8. Thepolymer library of claim 6, wherein said beads are contained in acombinatorial synthesis chamber.
 9. A polymer library comprisingpolymers selected from the group consisting of oligonucleotides andpeptides, wherein said library comprises at least three families ofpolymer pools attached to a substrate array, which polymers eachcomprise at least three monomers obtained from a basis set of monomers,where: the polymers in a first family are represented byX_(3P)X_(2P)X_(1P) wherein X₃ represents a first monomer position whichis occupied by a different monomer in each of the pools of polymers,X_(2P) represents a second monomer position which is occupied by amixture of monomers, and X_(1P) represents a third monomer positionwhich is occupied by a mixture of monomers; the polymers in a secondfamily are represented by X_(3P)X_(2P)X_(1P) wherein X_(3P) represents afirst monomer position which is occupied by a mixture of monomers, X₂represents a second monomer position which is occupied by a differentmonomer in each of the pools of polymers, and X_(1P) represents a thirdmonomer position which is occupied by a mixture of monomers; thepolymers in a third family are represented by X_(3P)X_(2P)X_(1P) whereinX_(3P) represents a first monomer position which is occupied by amixture of monomers, X_(2P) represents a second monomer position whichis occupied by a mixture of monomers, and X₁ represents a third monomerposition which is occupied by a different monomer in each of the poolsof polymers; wherein each pool is physically isolated from all the otherpools in the same family such that a polymer sequence of at least threemonomers which binds to a receptor can be identified by exposing thepools in the library to the receptor, whereby the pools in a familyreveal the identity of a monomer in a specific position of the polymersequence and simultaneous exposure of all the families in the library tothe receptor allows the determination of the identity of at least threeadjacent monomers in the sequence.
 10. The polymer library of claim 9,wherein said polymers comprise at least four monomers.
 11. The polymerlibrary of claim 9, further comprising a labeled receptor, and a meansfor identifying which of said families of polymers binds to said labeledreceptor.
 12. The polymer library of claim 11, wherein said receptor islabeled with a fluorescein label.
 13. The polymer library of claim 9,wherein each pool of polymers is bound to a predetermined region of asurface.